Method for inhibition of cell motility by sphingosine-1-phosphate, its derivatives and mimetics and method of synthesizing sphingosine-1-phosphate and its derivatives

ABSTRACT

A method of inhibiting tumor cell chemotactic and/or chemoinvasion motility comprising contacting the tumor cell with an inhibitory amount of an agent selected from the group consisting of sphingosine-1-phosphate, derivatives of sphingosine-1-phosphate and mimetics of the sphingosine-1-phosphate or of the derivatives. A method of inhibiting phagokinetic activity of tumor cells and neutrophils comprising contacting the cells with a phagokinetic inhibitory amount of an agent selected from the group consisting of sphingosine-1-phosphate, derivatives of sphingosine-1-phosphate, and mimetics of the sphingosine-1-phosphate or of the derivatives. A method of inhibiting tumor cell metastasis comprising administering to a host in need of treatment a metastasis inhibitory amount of an agent selected from the group consisting of sphingosine-1-phosphate, derivatives of sphingosine-1-phosphate, and mimetics of the sphingosine-1-phosphate or of the derivatives, and pharmaceutically acceptable salts of the agent. A method of inhibiting inflammation due to motility and invasion into blood vessel walls of neutrophils comprising administering to a host in need of treatment an inflammation inhibitory amount of an agent selected from the group consisting of sphingosine-1-phosphate, derivatives of sphingosine-1-phosphate, and mimetics of the sphingosine-1-phosphate or of the derivatives, and pharmaceutically acceptable salts of the agent. A method of preparing sphingosine-1-phosphate and its derivatives.

This is a divisional of application Ser. No. 07/863,179 filed Apr. 3,1992 U.S. Pat. No. 5,260,288.

FIELD OF THE INVENTION

The invention relates to compounds with profound effects on mammaliancell motility, methods of using the compounds and methods of chemicallysynthesizing the compounds.

BACKGROUND OF THE INVENTION

Sphingenine- and sphingosine-1-phosphate, collectively calledsphingosine-1-phosphate (SPN-1-P), have been known for many years asproducts of sphingosine (SPN) kinase {Stoffel W., Hoppe-Seyler's Z.Physiol. Chem., 354: 562, 1973; 354:1311 (1973); Stoffel W., et al,ibid, 355:61 (1974); 354:169 (1973); Louie D.D., et al, J. Biol. Chem.,251:4557 (1976)}. The reaction catalyzed by sphingosine (SPN) kinase isregarded as an initial step of sphingoid base degradation to yieldethanolamine-1-phosphate and a long-chain aldehyde (e.g., palmital) by apyridoxal phosphate-dependent lyase reaction (see FIG. 1). While SPN-1-Phas been recognized as an initial catabolic product of SPN, the realphysiological function of this compound has been unknown. SPN-dependentstimulation of mouse 3T3 cell growth has been shown to be independent ofthe protein kinase C (PKC) pathway {Zhang et al, J. Biol. Chem., 265:76(1990)}, and has been attributed to formation of SPN-1-P {Zhang et al,J. Cell Biol., 114:155 ( 1991)}. SPN-1-P may enhance cytoplasmic Ca²⁺release in analogy to the effect of inositol-1,4,5-triphosphate on Ca²⁺movement {Ghosh et al, Science, 248:1653 (1990)}. Although SPN-1-P wasassumed in these earlier studies to induce a cell-proliferative effectof 3T3 cells, particularly in the presence of epidermal growth factorand insulin {Zhang et al (1991)}, the physiological functional role ofSPN-1-P in cells has been unknown.

On the other hand, SPN-1-P is difficult to synthesize from chemicalreactions. B. Weiss {J. Am. Chem. Soc., 79:5553 (1957)} was able tosynthesize dihydrosphingosine-1-P (sphinganine-1-P) but notsphingenine-1-P. This effort to chemically synthesize SPN-1-P wasunsuccessful, probably because of the presence of multi-functionalgroups in SPN. The only reported method for preparation of SPN-1-P(mainly D-erythro isomer, but containing a small Mount of L-threoisomer) is by treatment of sphingosylphosphocholine with phospholipaseD, isolated from Streptomyces chromofuscus {van Veldhoven P. P.,Fogelsong R. J., Bell R. M., J. Lipid Res., 30:611 (1989)}.

SUMMARY OF THE INVENTION

The present inventors have found the SPN-1-P and its derivatives affectcell motility. Cell motility is an important parameter defining variouspathological processes such as inflammation, tumor invasion, andmetastasis.

Accordingly, one object of the invention is to provide a compound andits derivatives for inhibiting metastatic properties of malignant tumorcells, for controlling cell motility and for treating various disorderscharacterized by abnormal cell proliferation.

Another object of the invention is to provide a compound and itsderivatives for inhibiting inflammation due to motility of neutrophils.

A further object of the invention is to provide methods of preparing acompound and its derivatives which inhibit metastatic properties ofmalignant tumor cells and inflammation due to motility of neutrophils.

These and other objects have been achieved by providing a method ofinhibiting tumor cell chemotactic motility comprising contacting thetumor cells with an inhibitory amount of an agent selected from thegroup consisting of sphingosine-1-phosphate, derivatives ofsphingosine-1-phosphate and mimetics of the sphingosine-1-phosphate orthe derivatives.

The present invention also provides a method of inhibiting tumor cellchemoinvasion comprising contacting the tumor cells with an inhibitoryamount of an agent selected from the group consisting ofsphingosine-1-phosphate, derivatives of sphingosine-1-phosphate, andmimetics of the sphingosine-1-phosphate or the derivatives.

The present invention also provides a method of inhibiting phagokineticactivity of tumor cells and neutrophils comprising contacting the cellswith a phagokinetic inhibitory amount of an agent selected from thegroup consisting of sphingosine-1-phosphate, derivatives ofsphingosine-1-phosphate, and mimetics of the sphingosine-1-phosphate orthe derivatives.

The present invention additionally provides a method of inhibiting tumorcell metastasis comprising administering to a host in need of treatmenta metastasis inhibitory amount of an agent selected from the groupconsisting of sphingosine-1-phosphate, derivatives ofsphingosine-1-phosphate, and mimetics of the sphingosine-1-phosphate orthe derivatives, and pharmaceutically acceptable salts of the agent.

The present invention even further provides a method of inhibitinginflammation due to motility and invasion into blood vessel walls ofneutrophils comprising administering to a host in need of treatment aninflammation inhibitory amount of an agent selected from the groupconsisting of sphingosine-1-phosphate, derivatives ofsphingosine-1-phosphate and mimetics of the sphingosine-1-phosphate orthe derivatives, and pharmaceutically acceptable salts of the agent.

Finally, the present invention provides sphingosine-1-phosphate and itsderivatives essentially free of L-threo isomer as detected by NMRspectroscopy and a method for preparing this sphingosine-1-phosphate andits derivatives.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the metabolic relationships in synthesis and degradationof sphingolipids. All glycosphingolipids (except GalCer and itsderivatives) are synthesized through GlcCer, which is synthesized fromceramide (Cer) through UDP-Glc. Cer is degraded into fatty acids and SPN(route 1). SPN is degraded through phosphorylation into SPN-1-P throughSPN kinase (route 2), which is in turn degraded into phosphoethanolamineand palmital. SPN can also be converted into dimethylsphingosine (DMS)by transmethylation (route 3). Cer is converted to sphingomyelin bytransfer of phosphorylcholine from phosphatidylcholine.

FIG. 2 gives the structure of SPN-1-P and various synthetic derivativesof SPN-1-P.

FIGS. 3A-3I depict chemical synthesis of SPN-1-P and its variousderivatives.

FIGS. 4A and 4B are negative ion fast atom bombardment mass spectra(DMIX as matrix) of SPN-1-P made from sphingosylphosphocholine withphospholipase D (FIG. 4A) and of SPN-1-P chemically synthesized (FIG.4B).

FIGS. 5A-D, are portions of the ¹ H-NMR spectra (500 MHz) of SPN-1-Pmade from sphingosylphosphocholine with phospholipase D (FIGS. 5A and5B) and of SPN-1-P chemically synthesized (FIGS. 5C and 5D) The spectrawere taken in methyl-¹² C-d₃ -alcohol-d-acetic-d₃ -acid-d 8:2 (v/v).

FIG. 6 depicts a scheme for chemotactic cell motility and chemoinvasionassays.

FIG. 7 is a graph showing the linear relationship between cell numberand toluidine blue optical density for an assay detecting chemotacticcell motility or chemoinvasion.

FIG. 8 is a graph depicting data that demonstrates the rationale forselection of MATRI-GEL quantity coated on transwell polycarbonatemembrane. The ordinate represents the number of migrating cells(determined by toluidine blue absorbance), and the abscissa representsthe quantity of MATRI-GEL applied. Closed circles represent migrationdetermined after 20 hours, and open circles represent migrationdetermined after 72 hours. For 20 hour duration, maximal migration wasobserved when 1 μg MATRI-GEL was coated per well filter, so thisquantity was used for the chemotactic motility assay. For the 72 hourduration, no migration was observed when 20 μg MATRI-GEL per well wasapplied, but some migration occurred with 10 μg per well. Therefore, 10μg was used for the chemoinvasion assay.

FIG. 9 is a graph showing chemotactic motility of mouse B16/F1 cellsthrough a polycarbonate transwell membrane coated with 1 μg/wellMATRI-GEL after 20 hour incubation. The ordinate represents the percentof cell number migrated relative to control. The abscissa representsconcentration of SPN or SPN-derivative in conditioned medium (CM) addedto the lower chamber. SPN-1-P represents sphingosine-1-phosphate and TMSrepresents N,N,N-trimethylsphingosine.

FIG. 10 is a graph showing chemoinvasion of B16/F1 cells through apolycarbonate transwell membrane coated with 10 μg/well MATRI-GEL after70 hours incubation. The ordinate, abscissa and abbreviations are thesame as described for FIG. 9.

FIGS. 11A-F depict gold sol clearance patterns of B16/F1 cells for thephagokinetic assay. FIGS. 11D-F show areas cleared in the absence of orin the presence of various concentrations of SPN-1-P.

FIG. 11A: control cells in CM without SPN-1-P;

FIG. 11B: CM plus 1.0 μM SPN-1-P;

FIG. 11C: CM plus 0.1 μM SPN-1-P;

FIG. 11D: 0 μM SPN-1-P;

FIG. 11E: 0. 1 μM SPN-1-P;

FIG. 11F: 1.0 μM SPN-1-P.

FIGS. 12A and 12B depict the time-course uptake of ³ H-SPN (FIG. 12A)and ¹⁴ C-TMS (FIG. 12B) by B16/F1 cells. The ordinate represents %radioactivity taken up by B16/F1 cells and the abscissa represents timein hours.

FIGS. 13A-13C depict the time-course changes in labeling of various SPNderivatives after addition of ³ H-SPN to B16/F1 cells:

FIG. 13A: thin-layer chromatography (TLC) of lipids separated fromFolch's lower phase;

FIG. 13B: TLC of lipids separated from Folch's upper phase followed byincubation with ¹⁴ C-TMS and extraction;

FIG. 13C: TLC of lipids separated from Folch's upper phase.

Lane 1, 0 minute. Lane 2, 10 minutes. Lane 3, 30 minutes. Lane 4, 1hour. Lane 5, 2 hours. Lane 6, 4 hours. Lane 7, 20 hours. CER representsceramide; CMH represents ceramide monohexoside; PE representsphosphatidyl-ethanolamine; SM represents sphingomyelin; TMS representsN,N,N-trimethylsphingosine; SPN represents sphingosine; SPN-1-Prepresents sphingosine-1-phosphate; and ORG represents the origin.

DETAILED DESCRIPTION OF THE INVENTION

Herein, the present inventors provide a clear demonstration that SPN-1-Pinhibits cell motility of neutrophils and tumor cells, as determined bya phagokinetic track assay (on gold sol particle-coated solid phase) andan invasion assay (through a transwell chamber coated with anextracellular matrix). This inhibitory effect on SPN-1-P is shown to bemuch stronger than that for SPN, N,N-dimethylsphingosine (DMS), orN,N,N-trimethylsphingosine (TMS). Further, in striking contrast to DMSand TMS, SPN-1-P does not inhibit PKC. Therefore, the effect of SPN-1-Pon cell motility is independent of the PKC signaling pathway.

The present inventors have also determined how to make SPN-1-P and itsderivatives by chemical synthesis.

Thus, this invention deals with the chemical synthesis and use ofSPN-1-P, its derivatives, or mimetics as inhibitors of cell motility ingeneral, and their use in suppression of tumor cell metastasis andinflammatory processes, both of which are highly dependent on cellmotility. SPN-1-P is far less cytotoxic than SPN, DMS, or TMS, andtherefore is anticipated to be more useful for clinical application thanSPN, DMS, TMS or other SPN derivatives.

Methods Of Inhibiting Tumor Cell Chemotactic Motility and Tumor CellChemoinvasion

The present invention provides a method of inhibiting tumor cellchemotactic motility comprising contacting the tumor cells with aninhibitory amount of an agent selected from the group consisting ofSPN-1-P, derivatives of SPN-1-P and mimetics of the SPN-1-P or of thederivatives.

Additionally, the present invention provides a method of inhibitingtumor cell chemoinvasion comprising contacting the tumor cells with aninhibitory amount of an agent selected from the group consisting ofSPN-1-P, derivatives of SPN-1-P and mimetics of the SPN-1-P or of thederivatives.

The inhibitory amount of agent used in each method can readily bedetermined by the assays using transwell plates described below.

As a general guideline, an inhibitory amount of SPN-1-P sufficient toinhibit tumor cell chemotactic motility and chemoinvasion is from about10⁻⁸ M to about 10⁻⁷ M.

The assays for determining chemotactic cell motility and chemoinvasioncan be performed using transwell plates with a polycarbonate membranefilter (pore size 8 μm) (Costar Scientific, Cambridge, Mass.). Aliguots,e.g., 50 μl, of an aqueous solution of MATRI-GEL (CollaborativeResearch, Bedford, Mass.) containing SPN-1-P or other inhibitor (e.g.,20 μg/ml for chemotactic motility assay or 200 μg/ml for chemoinvasionassay), is added to each well and dried overnight. The filter is thenfitted onto the lower chamber plate. The lower chamber can containconditioned medium (CM) (i.e., medium used for splenic stromal cellculture, and containing motility factor secreted by these cells), e.g.,0.6 ml, with or without SPN-1-P or other inhibitor. To the upper chamberis added, e.g., about 100 μl, of cell suspension (5×10⁴ cells/ml forinvasion assay, 5×10⁵ cells/ml for motility assay), which is thenincubated in 5% CO₂ at 37° C. for 70-72 hours (invasion assay) or 20hours (motility assay). After incubation, cells remaining in the upperchamber are wiped off with a cotton swab, and cells which had migratedto the lower chamber side of the filter are fixed in methanol for 30seconds and stained with 0.05% toluidine blue. The filter is removed,the stain is solubilized in 10% acetic acid (e.g., 0.1 ml for invasionassay, 0.5 ml for motility assay), and color intensity (optical density)is quantitated by ELISA reading at 630 nm. A schematic summary of thisprocedure is shown in FIG. 6. Using SPN-1-P, a linear relationship wasobserved between cell number and toluidine blue optical density (FIG.7).

Method of Inhibiting Phagokinetic Activity of Tumor Cells andNeutrophils

The present invention also provides a method of inhibiting phagokineticactivity of tumor cells and neutrophils comprising contacting the cellswith a phagokinetic inhibitory amount of an agent selected from thegroup consisting of SPN-1-P, derivatives of SPN-1-P and mimetics of theSPN-1-P or of the derivatives.

The inhibitory amount of agent can readily be determined by assays knownin the art, such as the gold sol-coated plate assay described below.Using this assay, phagokinetic inhibitory amounts of SPN-1-P for tumorcells range from about 0.1 μM to about 1.0 μM, and phagokineticinhibitory amounts of SPN-1-P for neutrophils range from about 0.45 μMto about 4.5 μM.

Phagokinetic activity is measured by the ability of cells to ingestforeign particles while moving. Cell motility can be estimated as thearea of a phagokinetic track on gold sol particle-coated plates aspreviously described {Albrecht-Buehler, Cell, 11:395 (1977)}. A uniformcoating of gold particles is prepared on glass coverslips precoated withbovine serum albumin, and coverslips are rinsed repeatedly to removenon-adhering or loosely-adhering gold particles. Freshly-preparedneutrophils or tumor cells detached from culture are placed in a Petridish containing the gold sol-coated plate, and incubated for about 2hours (for human neutrophils) or about 18 hours (for tumor cells). Thecoverslips are fixed for 1 hour in a 4% formaldehyde solution inphosphate-buffered saline (PBS) and mounted on microscope slides. Thephagokinetic tracks are observed on a television connected to a lightmicroscope (Nikon, Tokyo, Japan). Tracks on the television aretransferred to translucent sheets, which are then photocopied.Phagokinetic activity is quantitated by cutting and weighing the sweptarea in the copy.

Method of Inhibiting Tumor Cell Metastasts and Method of InhibitingInflammation

The above-described assays establish that SPN-1-P adversely affectsmotility properties of tumor cells and neutrophils. SPN-1-P is clearlydemonstrated to have a strong inhibitory effect on motility of bothtypes of cells. Because the processes of tumor cell invasion andinflammation are dependent on the motility properties of tumor cells andneutrophils, respectively, SPN-1-P, its derivatives and mimetopes ofSPN-1-P or its derivatives are expected to be useful in the suppressionof tumor metastasis and in the inflammatory process.

For comparison purposes, the same test cells used in the above-describedassays were also exposed to numerous other sphingolipids and SPN-1-Pdemonstrated unexpectedly superior inhibition of both chemotacticmotility and chemoinvasion as shown in Table IV in Example II below.

In addition, the inhibitory effect of SPN-1-P on chemotactic motilitythrough MATRI-GEL-coated polycarbonate filters of mouse melanoma B16/F1and B16/F10 cells, mouse Balb/c 3T3 fibroblasts and human fibrosarcomaHT1080 cells was compared. The results, as shown in Table V in ExampleII below, establish that susceptibility of B16/F1 and B16/F10 cells tosphingosine-1-phosphate was high, whereas as that of human fibrosarcomaHT1080 cells was low.

Also, for comparison purposes, B16/F10 melanoma cells were exposed toSPN and TMS in the assay to determine phagokinetic activity. As shown inTable VI in Example III below, addition of SPN or TMS to the culturemedium reduced the area cleared by tumor cells. In particular, however,the average cleared area was greatly reduced when SPN-1-P was added at aconcentration of 1.0 or even 0.1 μM.

Phagokinetic activity of human neutrophils was, for comparison purposes,also determined using SPN, TMS, phosphoethanolamine, and ceramide. Asshown in Table VII in Example III below, the reduction in phagokineticactivity of human neutrophils was most striking for SPN-1-P and TMS.

Effects of SPN derivatives on protein kinase C activity and cell growthof B16/F1 cells was also investigated. SPN-1-P had no inhibitory effecton PKC activity of B16/F1 cells, even at 75 μM, whereas both SPN and TMSshowed a strong inhibitory effect at this concentration (Table I). TMSand SPN showed, respectively, a strong and a moderate growth-inhibitoryeffect on B16/F1 cells at 10 μM, whereas SPN-1-P showed nogrowth-inhibitory effect at this concentration (Table II). Toxicity ofthese compounds to B16/F1 cells and human neutrophils was also examinedusing a trypan blue exclusion assay after 1 hour incubation with thecompounds. SPN-1-P showed weak toxicity against both types of cells at45-50 μM, whereas SPN was very toxic at this concentration (Table III).

                  TABLE I                                                         ______________________________________                                        Effect of SPN derivatives on                                                  PKC activity of B16/F1 melanoma cells.                                        Compound     Conc. (μM)                                                                            % PKC activity                                        ______________________________________                                        Control                 100 ± 11                                           SPN          75         34 ± 7                                             SPN-1-P      75         108 ± 32                                                        10         121 ± 10                                           TMS          75         16 ± 3                                             ______________________________________                                         *Mean ± S.E. (n = 3). For controls (defined as 100%), PKC activity was     33225 cpm per tube per 20 min, as measured by .sup.32 P incorporation int     histone IIIS.                                                            

                  TABLE II                                                        ______________________________________                                        Effect of SPN derivatives on                                                  growth of B16/F1 melanoma cells.                                              Compound      Conc. (μM)                                                                            % Growth                                             ______________________________________                                        Control                  100 ± 4                                           SPN           10          78 ± 4*                                                        5           87 ± 10                                                        1          101 ± 1                                           SPN-1-P       10          87 ± 10                                                        5          96 ± 2                                                          1          105 ± 10                                                        0.1        97 ± 5                                                          0.01       103 ± 10                                          TMS           10         11 ± 1                                                          5          77 ± 8                                                          1          88 ± 7                                            N-acetyl-SPN  10         102 ± 4                                           ______________________________________                                         *Mean ± S.E. (n = 3). For controls (defined as 100%), cell number was      5.5 ± 0.2 × 10.sup.5 /dish. 10.sup.5 cells were seeded and           cultured on a 35 mm plastic dish in Dulbecco's modified Eagle's medium        containing 2% fetal bovine serum in the presence or absence of SPN            derivatives. 48 hr later, cells were counted.                            

                  TABLE III                                                       ______________________________________                                        Toxicity of SPN derivatives on                                                B16/F1 melanoma cells and human neutrophils.                                  Cell    Compound   Conc. (μM)                                                                             Cell viability (%)                             ______________________________________                                        B16/F1  control                99                                                     SPN        50          21                                                     SPN-1-P    50          72                                             neutrophil                                                                            control                99                                                     SPN        45           5                                                     SPN-1-P    45          98                                             ______________________________________                                    

In addition, the uptake and metabolic conversion of SPN vs. TMS wasinvestigated. Both ³ H-labeled SPN and ¹⁴ C-labeled TMS were rapidlyincorporated into B16/F1 cells (FIGS. 12A and 12B). However, only SPNwas rapidly converted into SPN-1-P and ceramide (Cer) (FIG. 13). Thiswas clearly demonstrated when cells were incubated with ³ H-SPN in thepresence of D-PDMP, which inhibits conversion of Cer into GlcCer andother glycosphingolipids. Rapid conversion of SPN intosphingosine-1-phosphate is clearly indicated by the appearance of bandscorresponding to SPN-1-P prior to conversion into Cer. The SPN-1-P peakappeared after 10 minutes incubation, whereas the Cer peak appearedafter 1 hour incubation. In contrast, although ¹⁴ C-TMS was rapidlytaken up by cells, the band corresponding to TMS was unchangedregardless of incubation time (FIG. 13). These findings suggest thatinhibitory effects on cell motility and invasion are due to rapidconversion of SPN into SPN-1-P.

Accordingly, the present invention provides a method of inhibiting tumorcell metastasis comprising administering to a host in need of treatmenta metastasis inhibitory amount of an agent selected from the groupconsisting of SPN-1-P, derivatives of SPN-1-P and mimetics of theSPN-1-P or of the derivatives, and pharmaceutically acceptable saltsthereof.

The present invention also provides a method of inhibiting inflammationdue to motility of neutrophils comprising administering to a host inneed of treatment an inflammation inhibitory amount of an agent selectedfrom the group consisting of SPN-1-P, derivatives of SPN-1-P, andmimetics of the SPN-1-P or of the derivatives, and pharmaceuticallyacceptable salts thereof.

A specific use of the method of inhibiting tumor cell metastasisincludes treatment of malignancies. The method of inhibitinginflammation is applicable to any inflammation which is due to motilityand invasion into blood vessel walls of neutrophils.

The inhibitory effective amount of SPN-1-P or other inhibitor can bedetermined using art-recognized methods, such as by establishing doseresponse curves in suitable animal models and extrapolating to human;extrapolating from suitable in vitro data, for example, as describedherein; or by determining effectiveness in clinical trials.

Suitable doses of SPN-1-P or other inhibitor according to this inventiondepend upon the particular medical application, such as the severity ofthe disease, the weight of the individual, age of the individual,half-life in circulation, etc., and can be determined readily by theskilled artisan. The number of doses, daily dosage and course oftreatment may vary from individual to individual.

SPN-1-P and other inhibitors can be administered in a variety of wayssuch as orally, parenterally and topically. Suitable pharmaceuticallyacceptable carriers, diluents or excipients which can be combined withSPN-1-P and other inhibitors for administration depend upon theparticular medical use and can be determined readily by the skilledartisan.

The SPN-1-P or other inhibitors with or without carrier can take avariety of forms, such as tablets, capsules, bulk or unit dose powdersor granules; may be contained with liposomes; or may be formulated intosolutions, emulsions, suspensions, ointments, pastes, creams, gels,foams or Jellies. Parenteral dosage forms include solutions, suspensionsand the like.

Additionally, a variety of art-recognized excipients, diluents, fillers,etc., are likely to be included in the dosage forms. Such subsidiaryingredients include disintegrants, binders, lubricants, surfactants,emulsifiers, buffers, moisturizers, solubilizers and preservatives. Theartisan can configure the appropriate formulation comprising inhibitorand seeking guidance from numerous authorities and references such as"Goodman & Gilman's, The Pharmaceutical Basis of Therapeutics" (6th Ed.,Goodman et al, MacMillan Publ. Co., N.Y. 1980).

In body sites that are characterized by continual cell growth or thatrequire cell growth inhibition because of dysfunction and that arerelatively inaccessible, SPN-1-P and other inhibitors can beadministered in a suitable fashion to ensure effective localconcentrations. For example, the inhibitors may be injected in a depotor adjuvant, carried in a surgically situated implant or reservoir thatslowly releases a fixed amount of inhibitor over a period of time or maybe complexed to recognition molecules with the capability of binding tothe site presenting with abnormal cell growth. An example of such acontemplated scenario is a recognition molecule that is an antibody withbinding specificity for a bone marrow specific antigen wherein themarrow-specific antibody is complexed to SPN-1-P or other inhibitor, thecomplex being administered to a patient with leukemia.

Synthesis of Sphingosine-1-Phosphate and Its Derivatives

Various sphingosine (SPN) derivatives can be synthesized chemically asshown in FIG. 2 and FIGS. 3A, 3B, and 3C. These includesphingosine-1-phosphate (SPN-1-P): compound 1',N,N-dimethylsphingosine-l-phosphate {DMS-i-P (2)},N,N,N-trimethylsphingosine-1-phosphate {TMS-1-P (3)}, N-acetyl andN-acylsphingosine-1-phosphate {N-acetyl and N-acyl-SPN-1-P (4)},sphingosine-1,3-diphosphate {SPN-1,3-diphosphate (5)},sphingosine-3-phosphate {SPN-3-P (6)}, sphingosine-1-thiophosphate{SPN-1-S-P (7)}, N,N-dimethylsphingosine-1-thiophosphate {DMS-1-S-P(8)}, and N,N,N-trimethylsphingosine-1-thiophosphate {TMS-1-S-P (9)}.The synthesis methods that are described below are conventional in theart and can be readily practiced by the skilled artisan.

Synthesis of Sphingosine-1-Phosphate (Compound 1)

FIG. 3A summarizes the new procedure for synthesis of SPN-1-P (1),starting with the protected SPN (1') prepared from previously-knownprocedures {Garmer P., Park J. M., J. Org. Chem., 52:2361 (1987); HeroldP., Helvetica Chimica Acta, 71:354 (1988); Radunz H. E., Devant R. M.,Eiermann V., Liebigs Ann. Chem., 1988:1103 (1988)}. In compound 1', X isa protecting group such as N-tert-butyloxycarbonyl (t-Boc).2-N-X-3-O-pivaloyl-D-erythro-SPN (3') is prepared by esterification ofC-3 OH group of compound 1', for example with pivaloyl chloride in drypyridine, to give compound 2', followed by selective deprotection of theprimary hydroxyl group, for example with p-toluenesulfonic acid (p-TsOH)in methanol (MeOH). Phosphorylation of the primary hydroxyl group ofcompound 3', for example with phosphorus oxychloride in the presence oftriethylamine and CH₂ Cl₂ (distilled from CaH₂) followed by hydrolysis,for example with 1N HCl in CHCl₃, yields 2-N-X-3-O-pivaloyl-D-erythro-SPN-1-P (compound 4'). Deprotection of the C-3 OHgroup (e.g., with nButN⁺ OH-[aq]/dioxane) and the amino group (e.g.,with TFA/CH₂ Cl₂) respectively gives the desired SPN-1-P (Compound 1).This synthetic product can be proven to be identical to that derivedfrom sphingosylphosphocholine in the ¹ H-NMR spectrum (500 MHz) and massspectrum (negative FAB, DMIX as matrix), which are shown in FIGS. 4 and5. The small difference in NMR spectrum reflects the fact thatenzymatically-synthesized SPN-1-P contains a small amount of L-threoisomer, whereas chemically-synthesized SPN-1-P does not contain anydetectable amount of L-threo isomer. Thus, chemically-synthesizedSPN-1-P, according to the present invention, is essentially free ofL-threo isomer as detected by NMR spectroscopy.

Synthesis of N,N-Dimethylsphingosine-1-Phosphate (Compound 2)

Compound 3' is treated, e.g., with trifluoroacetic acid (TFA) and CHCl₂,to eliminate the protecting moiety X, and then reductive methylation iscarried out, e.g., in the presence of 37% CH₂ O and NaCNBH₃ in sodiumacetate aqueous buffer, resulting in compound 3^(a). Compound 3^(a) isthen phosphorylated, e.g., with POCl₃ in triethylamine (Et₃ N) and CH₂Cl₂, and the Cl atom is replaced with an OH group by treatment, e.g.,with 1N HCl in CHCl₃, resulting in compound 3^(b). The pivaloyl group atthe C-3 OH is eliminated, e.g., by treatment in tetrabutylammoniumhydroxide (nBu₄ N⁺ OH⁻) in aqueous dioxane, resulting in compound 2.

Synthesis of N,N,N-Trimethylsphingosine-1-Phosphate (Compound 3)

Compound 3^(a) is permethylated, e.g., with CH₃ I and NaHCO₃ in CHCl₃,followed by DOWEX 1×2 (Cl⁻) treatment to give compound 3^(h). Next, theC-1 OH is phosphorylated, e.g., with POCl₃ in Et₃ n and CH₂ Cl₂,followed by replacement of Cl by an OH group by treatment, e.g., with 1NHCl and CHCl₃. Next, the pivaloyl group is eliminated, e.g., bytreatment in the presence of nBu₄ N⁺ OH⁻ in aqueous dioxane, resultingin compound 3.

Synthesis of Sphingosine-1-Thiophosphate (Compound 7)

Compound 3' is treated with tosyl chloride (TsCl) in Et₃ N and CH₂ Cl₂,followed by treatment with potassium thioacetate inN,N-dimethylformamide (DMF) to yield compound 3^(c). The acetyl group isremoved by treatment with NaBH₄ in ethanol (EtOH) and CH₂ Cl₂. Next, theSH group is phosphorylated, e.g., with POCl₃ in Et₃ N and CH₂ Cl₂followed by treatment in 1N HCl in CHCl₃, to yield compound 3^(d).Compound 3^(d) is treated, e.g., with nBu₄ N⁺ OH⁻ in aqueous dioxane toeliminate the pivaloyl group at the C-3 OH. Next, X is eliminated, e.g.,with TFA in CH₂ Cl₂, to yield compound 7.

Synthesis of N,N-Dimethyl-Sphingosine-1-Thiophosphate (Compound 8)

Compound 3^(c) is treated, e.g., with TFA in CH₂ Cl₂ to eliminate theprotecting group X (e.g., t-Boc), and reductive methylation is carriedout, e.g., with 37% CH₂ O and NaCNBH₃ in aqueous acetate buffer, toreplace the amino group with an N-dimethyl group, yielding compound3^(e). Compound 3^(e) is treated with NaBH₄ in EtOH and CH₂ Cl₂,followed by phosphorylation, e.g., with POCl₃ in Et₃ N and CH₂ Cl₂, andtreatment with 1N HCl in CHCl₃ to yield compound 3^(f). Compound 3^(f)is treated, e.g., with nBu₄ N⁺ OH⁻ in aqueous dioxane, to eliminate thepivaloyl group at the C-3 OH, yielding compound 8.

Synthesis of N,N,N-Trimethylsphingosine-1-Thiophosphate (Compound 9)

Compound 3^(e) is treated, e.g., by Purdie permethylation with CH₃ I,NaHCO₃, and CHCl₃, followed by treatment with DOWEX 1×2 (Cl⁻), to yieldcompound 3^(g). Compound 3^(g) is treated, e.g., with NaBH₄, EtOH, andCH₂ Cl₂, to create an SH group at the 1-position of sphingosine. Next,the SH group is phosphorylated, e.g., with POCl₃ in Et₃ N and CH₂ Cl₂,followed by replacement of Cl with an OH group, e.g., by treatment with1N HCl and CHCl₃, followed by treatment, e.g., with nBu₄ N⁺ OH⁻ inaqueous dioxane, to eliminate the pivaloyl group at the C-3 OH, yieldingcompound 9.

Synthesis of N-Acetylsphingosine-1-Phosphate (Compound 4)

Compound 3' is treated, e.g., with TFA in CH₂ Cl₂, to eliminate theprotecting group X (e.g., t-Boc), and then treated, e.g., with CH₃(CH₂)_(n) COCl (n=0 to 22) in 50% K₂ CO₃ {in aqueous tetrahydrofuran(THF)} to acetylate or acylate the ammonium group to yield compound3^(i). Compound 3^(i) is treated, e.g., with POCl₃ in Et₃ N and CH₂ Cl₂and then with 1N HCl in CHCl₃, to phosphorylate the C-1 OH group toyield compound 3^(j). Compound 3^(j) is then treated, e.g., with nBu₄ N⁺OH⁻ in aqueous dioxane to eliminate the pivaloyl group at the C-3 OHyielding compound 4.

Synthesis of Sphingosine-1,3-Diphosphate (Compound 5)

Compound 1' is treated, e.g., with AMBERLYST number 15 in CH₃ OH, toselectively deprotect the C-1 OH group in order to yield compound 3^(k).Compound 3^(k) is then treated with (C₂ H₅ S)₂ PCl in dimethyl anilineand ethyl acetate (EtOAc) in order to form a P(SC₂ H₅)₂ group at the C-3hydroxyl and at the C-1 hydroxyl to give compound 3^(l). Compound 3^(l)is then treated, e.g., with I₂ in CH₃ OH and then with TFA in CH₂ Cl₂ inorder to phosphorylate the C-1 OH and the C-3 OH and deprotect the aminogroup to give compound 5.

Synthesis of Sphingosine-3-Phosphate (Compound 6)

Compound 1' is treated with (C₂ H₅ S)₂ PCl in dimethyl aniline and EtOActo form a P(SC₂ H₅)₂ group at the C-3 hydroxyl to give compound 3^(m).Compound 3^(m) is then treated, e.g., with HCl in dioxane and then withI₂ in CH₃ OH, in order to phosphorylate the C-3 hydroxyl and deprotectthe C-1 OH and the amino group to give compound 6.

The invention will now be described by reference to specific exampleswhich are not meant to be limiting. Unless otherwise specified, allpercents, ratios, etc., are by volume.

EXAMPLES Example I Preparation of Sphingosine-1-Phosphate

Sphingosine-1-phosphate (SPN-1-P) was synthesized both enzymatically andchemically.

Enzymatic synthesis was achieved through degradation ofsphingosylphosphocholine by phospholipase D as previously described{Veldhoven et al, J. Lipid Res., 30:611 (1989)}.

FIG. 3A summarizes the procedure for chemical synthesis of SPN-1-P,starting with the protected SPN-1 (1') prepared from previously-knownprocedures {Garmer P., Park J. M., J. Org. Chem., 52:2361 (1987); HeroldP., Helvetica Chimica Acta, 71:354 (1988); Radunz H. E., Devant R. M.,Eiermann V., Liebigs Ann. Chem., 1988:1103 (1988)}. For purposes of thisexample, the protected SPN-1 was protected with N-tert-butyloxycarbonyl(t-Boc). Synthesis of the compound 2', 0.22 g (94%), as a colorless oil,was accomplished by esterification of the C-3 OH group of the protectedsphingosine 1' (0.20 g, 0.46 mmol) with pivaloyl chloride (1.0 ml, 8.1mmol) in 5 ml of dry pyridine at 25° C. for 4h, which was purified bysilica-gel chromatography (EtOAc/hexane, (1:8 v:v)). Selectivedeprotection of the C-1 OH group of 2' (0.21 g, 0.40 mmol) by treatmentwith p-toluenesulfonic acid (˜100 mg), in 10 ml of methanol at 25° C.for 5 h afforded 2-N-t-Boc-3-O-pivaloyl-D-erythro-SPN (3'), 0.135 g(70%), as a colorless oil (silica gel chromatography, EtOAc/hexane (1:4v:v)). Phosphorylation of the C-1 hydroxyl group of compound 3 (14 mg,0.029 mmol) with phosphorus oxychloride (26 μl, 0.27 mmol) in thepresence of triethylamine (43 μl, 0.3 mmol) and 0.5 ml of CH₂ Cl₂(distilled from CaH₂) at 25° for 2h followed by hydrolysis with 1 ml of1N HCl and 1 ml of CHCl₃ (25° C., 1.5 h) yielded 12.9 mg (80%) of2-N-t-Boc-3-O-pivaloyl-D-erythro-SPN-1-P (compound 4') (silica gelchromatography with CH₂ Cl₂ /CH₃ OH/AcOH, 6:1:0.2, v:v:v). Finally,deprotection of the C-3 OH group of the compound 4' (12.9 mg, 0.023mmol) ((1) 35 drops of 40 wt % nBu₄ N⁺ OH⁻ (aq.)/3 ml of dioxane, 4h,25° C.; (2) AMBERLITE IR-120, H₂ O) followed by removal of the aminoprotecting group (8 ml 50% TFA/CH₂ Cl₂, 0.5 h, 25° C.) gave the desiredSPN-1-P, 10 mg (77%), as a white solid (HOAc as a counterion), which waspurified by silica gel chromatography (nBuOH/H₂ O/AcOH, 6:1:1, v:v:v).

This synthetic product proved identical to that derived fromsphingosylphosphocholine in the ¹ H-NMR spectrum (500 MHz) and massspectrum (negative FAB, DMIX as matrix), which are shown in FIGS. 4 and5.

FIGS. 4A and 4B show negative ion fast atom bombardment mass spectra(DMIX as matrix) of SPN-1-P made from sphingosylphosphocholine withphospholipase D (FIG. 4A) and of SPN-1-P chemically synthesized (FIG.4B).

FIGS. 5A-D, are portions of the ¹ H-NMR spectra (500 MHz) of SPN-1-Pmade from sphingosylphosphocholine with phospholipase D (FIGS. 5A and5B) and of SPN-1-P chemically synthesized (FIGS. 5C and 5D). The spectrawere taken in methyl-¹² C-d₃ -alcohol-d-acetic-d₃ -acid-d 8:2 (v/v).

The small difference in NMR spectrum reflects the fact thatenzymatically-synthesized SPN-1-P contains a small amount of L-threoisomer, whereas chemically-synthesized SPN-1-P does not contain anydetectable amount of L-threo isomer.

Example II Assays for Chemotactic Cell Motility and Chemoinvasion usingTranswell Plates

Assays were performed using transwell plates with polycarbonate membranefilters (pore size 8 μm) (Costar Scientific, Cambridge, Mass.). 50 μlaliguots of an aqueous solution of MATRI-GEL (Collaborative Research,Bedford, Mass.) containing 20 μg/ml (for chemotactic motility assay) or200 μg/ml (for chemoinvasion assay) were added to each well and driedovernight. The filter was fitted onto the lower chamber plate. The lowerchamber contained 0.6 ml conditioned medium (CM) (i.e., medium used forsplenic stromal cell culture, and containing motility factor secreted bythese cells) with or without the suspected inhibitor. To the upperchamber was added 100 μl of cell suspension (5×10⁴ cells/ml for invasionassay, 5×10⁵ cells/ml for motility assay), which was then incubated in5% CO₂ at 37° C. for 70-72 hours (invasion assay) or 20 hours (motilityassay). After incubation, cells remaining in the upper chamber werewiped off with a cotton swab, and cells which had migrated to the lowerchamber side of the filter were fixed in methanol for 30 seconds andstained with 0.05% toluidine blue. The filter was removed, the stain wassolubilized in 10% acetic acid (0.1 ml for invasion assay, 0.5 ml formotility assay), and color intensity (optical density) was quantitatedby ELISA reading at 630 nm. A schematic summary of this procedure isshown in FIG. 6. A linear relationship was observed between cell numberand toluidine blue optical density (FIG. 7.).

Inhibition of Chemotactic Cell Motility by SPN-1-P

In experiments with different quantities of MATRI-GEL, cell migrationthrough transwell filter was maximal with 1 μg/well was applied, andwhen CM was used (FIG. 8). Therefore, chemotactic cell motility, asaffected by various SPN derivatives, was assayed under these conditions.

The results for chemotactic motility of mouse melanoma B16/F1 cells areshown in FIG. 9. In FIG. 9, the ordinate represents the percent of cellnumber migrated relative to control, and the abscissa representsconcentration of SPN or SPN-derivative in CM added to the lower chamber.The results establish that the motility for mouse melanoma B16/F1 cellswas inhibited most strongly by SPN-1-P, followed by SPN and TMS.Motility (i.e., penetration through the MATRI-GEL-coated filter) was100% blocked by 10⁻⁷ M SPN-1-P, and 90% blocked by 10⁻⁸ M SPN-1-P. Bothenzymatically- and chemically-synthesized SPN-1-P showed the samedose-dependent inhibitory effect on cell motility. A much higherconcentration (10⁻⁵ M) of SPN was required for 100% blocking. 10⁻⁵ M TMSproduced only weak inhibition of motility. The higher effectiveness ofSPN compared to TMS is due to the fact that SPN can be converted toSPN-1-P, whereas TMS cannot be phosphorylated.

Inhibition of Chemoinvasion

Chemoinvasion was measured by the ability of tumor cells in CM (asdescribed above) to migrate through a thick layer of MATRI-GEL during aprolonged incubation period (70 hours). This property is distinct fromchemotactic cell motility, which involves a much shorter incubationperiod (20 hours) and a thin layer of MATRI-GEL. For the chemoinvasionassay, 10 μg of MATRI-GEL was applied to a polycarbonate transwellfilter and migration was observed following 70 hours incubation (basedon results shown in FIG. 8).

The results are shown in FIG. 10. In FIG. 10, the ordinate representsthe percent of cell number migrated relative to control, and theabscissa represents the concentration of SPN or SPN-derivative (in CM)added to the lower chamber. The results show that under theseconditions, invasion of B16/F1 cells was strongly inhibited by 10⁻⁸ or10⁻⁷ M SPN-1-P, whereas SPN and TMS had a weaker effect. The differencein effect for SPN-1-P vs. SPN or TMS was not as pronounced as formotility.

Comparative effects of various sphingolipids on chemotactic cellmotility and chemoinvasion of B16/F1 cells are summarized in Table IV.Effect of SPN-1-P on motility of various cells is shown in Table V.Susceptibility of B16/F1 and B16/F10 cells to SPN-1-P was high, whereasthat of human fibrosarcoma HT1080 cells was low.

                  TABLE IV                                                        ______________________________________                                        Comparative effects of sphingolipids on chemotactic motility                  and chemoinvasion of B16/F1 melanoma cells.                                   Sphingolipid      % motility                                                                              % invasion                                        ______________________________________                                        control           100 ± 9                                                                              100 ± 20                                       SPN                78 ± 11*                                                                            16 ± 7                                         SPN-1-P            5 ± 1 12 ± 4                                         phosphoethanolamine                                                                              86 ± 20                                                                             160 ± 57                                       ethanolamine       85 ± 13                                                                             140 ± 41                                       phosphatidylethanolamine                                                                        107 ± 18                                                                             104 ± 37                                       Cer (Sigma, type III)                                                                           101 ± 26                                                 8-Cer             125 ± 15                                                                              82 ± 13                                       N-acetyl-SPN       99 ± 16                                                                              96 ± 14                                       CMH               162 ± 29                                                                             178 ± 70                                       GM3               140 ± 26                                                                             127 ± 69                                       sphingomyelin      82 ± 11                                                                             138 ± 18                                       sulf-SPN          114 ± 36                                                                             160 ± 23                                       Cer-1-P           136 ± 12                                                                              37 ± 18                                       TMS               100 ± 19                                                                             75 ± 8                                         ______________________________________                                         *Mean ± S.E. of percent relative to control (n = 3 or 4).             

                  TABLE V                                                         ______________________________________                                        Effect of SPN-1-P on chemotactic motility through                             MATRI-GEL-coated polycarbonate filter of mouse melanoma                       B16/F1 and B16/F10 cells, mouse Balb/c 3T3 fibroblasts,                       and human fibrosarcoma HT1080 cells.                                          SPN-1-P  Relative Motility                                                    dose (μM)                                                                           F1        F10        3T3    HT1080                                   ______________________________________                                        5.0      --        --         11 ± 1                                                                            64 ± 4                                1.0       8 ± 2*                                                                              4 ± 1   40 ± 1                                                                            105 ± 14                              0.1      6 ± 2  4 ± 2   101 ± 10                                                                          115 ± 35                              0.01     12 ± 7 10 ± 4  125 ± 5                                                                           100 ± 30                              0.001    82 ± 44                                                                              96 ± 21 119 ± 9                                                                           100 ± 21                              control  100 ± 9                                                                              100 ± 16                                                                              100 ± 10                                                                          100 ± 10                              ______________________________________                                         *Mean ± S.E. of percent relative to control (n = 4). Actual O.D. value     of controls (defined as 100%) were 0.114 (F1), 0.199 (F10), 0.322 (3T3),      and 0.147 (HT1080). 6 × 10.sup.4 cells were placed on a transwell       filter coated with 1.0 μg MATRIGEL in the upper chamber, and cultured      for 18 hours in the presence of CM and SPN1-P in the lower chamber.      

Example III Phagokinetic Assay using Gold Sol-Coated Plates

Cell motility was estimated as the area of phagokinetic track on goldsol particle-coated plates as previously described {Albrecht-Buehler,Cell, 11:395 (1977)}. A uniform coating of gold particles was preparedon glass coverslips precoated with bovine serum albumin, and thecoverslips were rinsed repeatedly to remove non-adhering orloosely-adhering gold particles. Freshly-prepared neutrophils or tumorcells detached from culture were placed in a Petri dish containing thegold sol-coated plate, and incubated for 2 hours (for human neutrophils)or 18 hours (for tumor cells). The coverslips were fixed for 1 hour in4% formaldehyde solution in phosphate-buffered saline (PBS) and mountedon microscope slides. The phagokinetic tracks were observed on atelevision connected to a light microscope (Nikon, Tokyo, Japan). Trackson the television were transferred to translucent sheets, which werethen photocopied. Phagokinetic activity was quantitated by cutting andweighing the swept area in the copy.

The results for inhibition of phagokinetic activity of tumor cells bySPN-1-P are shown in FIGS. 11A-F. FIGS. 11A-F show the gold solclearance patterns of B16/F1 cells. FIG. 11A: control cells in CMwithout SPN-1-P; FIG. 11B: CM plus 1.0 μM SPN-1-P; FIG. 11C: CM plus 0.1μM SPN-1-P; FIGS. 11D-F show areas cleared in the absence of or in thepresence of various concentrations of SPN-1-P: FIG. 11D, 0 μM SPN-1-P;FIG. 11E, 0.1 μM SPN-1-P; FIG. 11F, 1.0 μM SPN-1-P.

The results show that addition of SPN or its derivatives to the culturemedium reduced the area cleared by tumor cells. In particular, averagecleared area was greatly reduced when SPN-1-P was added at aconcentration of 1.0 or even 0.1 μM. The comparative effects of variousSPN derivatives on B16/F10 phagokinetic activity are summarized in TableVI.

                  TABLE VI                                                        ______________________________________                                        Effect of SPN, SPN-1-P, and TMS on phagokinetic                               activity of B16/F10 melanoma cells.                                           Com-   Conc.    Cleared area                                                                             less control                                                                           % of control                              pound  (μM)  (× 10.sup.3 μm.sup.2)                                                           (CM-) value                                                                            (CM+) value                               ______________________________________                                        Control         2.1 ± 0.9*                                                                            0                                                  (CM-)                                                                         Control         6.9 ± 3.4                                                                             4.8      100                                       (CM+)                                                                         SPN    1.0      2.5 ± 1.0.sup.a                                                                       0.4       8                                        SPN-1-P                                                                              1.0      2.4 ± 1.1.sup.a                                                                       0.3       6                                               0.1      3.4 ± 1.3.sup.a                                                                       1.3      27                                                0.01    4.1 ± 1.4.sup.a                                                                       2.0      42                                                 0.0001 6.0 ± 2.7                                                                             3.9      81                                        TMS    1.0      5.6 ± 2.7.sup.b                                                                       3.5      73                                        ______________________________________                                         *Mean ± S.D. (n > 50). 10.sup.3 B16/F10 cells were seeded on a             coverslip precoated with gold sol particles in the presence or absence of     SPN derivative. 18 hours later, the cleared area was estimated as             described in the text.                                                        .sup.a p < 0.0001,                                                            .sup.b p < 0.05 compared to control. Using B16/F1 cells, similar results      were obtained (data not shown).                                          

The effects of SPN derivatives on myeloid cell phagokinesis are shown inTable VII. As seen from the data in Table VII, reduction of phagokineticactivity of human neutrophils was most striking for SPN-1-P and TMS.

                  TABLE VII                                                       ______________________________________                                        Effect of SPN, SPN-1-P, and TMS on phagokinetic                               activity of human neutrophils.                                                Compound Conc. (μM)                                                                            n*      Cleared area (× 10.sup.3                    ______________________________________                                                                    μm.sup.2)                                      Control             141      6.3 ± 2.3**                                   SPN      0.45       81      5.2 ± 2.0.sup.a                                TMS      0.45       94      5.4 ± 2.7.sup.c                                         1.0        100     3.6 ± 1.5.sup.a                                SPN-1-P  0.45       80      5.5 ± 2.7.sup.c                                         1.0        74      5.4 ± 2.4.sup.b                                         4.5        123     3.5 ± 1.4.sup.a                                phospho- 4.5        70      5.9 ± 2.5                                      ethanolamine                                                                  Cer      4.5        75      5.7 ± 3.0                                      ______________________________________                                         *n, number of neutrophils examined.                                           **mean ± S.D. Freshlyprepared neutrophils (1 × 10.sup.4              cell/plate) were seeded on a coverslip precoated with gold sol particles      in the presence or absence of test compound. 2 hours later, incubation wa     terminated by adding 200 μl of 10% formaldehyde and the cleared area       was estimated as described in the text.                                       .sup.a p < 0.001,                                                             .sup.b p < 0.025,                                                             .sup.c p < 0.05 compared to control.                                     

While the invention has been described in detail above with reference toa preferred embodiment, various modifications within the scope andspirit of the invention will be apparent to people of working skill inthis technological field.

What is claimed:
 1. A method for preparing sphingosine-1-phosphate essentially free of L-threo isomer comprising(1) protecting the C1 and C3 hydroxyl groups of sphingosine (SPN); (2) deprotecting the C1 hydroxyl group of SPN; (3) phosphorylating the C1 hydroxyl group of SPN; to produce sphingosine-1-phosphate essentially free of L-threo isomer; and (4) deprotecting the C3 hydroxyl group of SPN.
 2. A method for chemically synthesizing sphingosine-1-phosphate comprising:(A) esterifying the C-3 hydroxyl group of compound 1' to give compound 2' ##STR1## wherein X represents a moiety that protects the amino group formed in step (B), (B) selectively deprotecting the C-1 hydroxyl group of said compound 2' to give compound 3' ##STR2## (C) phosphorylating the C-1 hydroxyl group of said compound 3', followed by, hydrolyzing to give compound 4' ##STR3## (D) deprotecting the C-3 hydroxyl group and the amino group, respectively, of said compound 4' to give compound 1, which is said sphingosine-1-phosphate ##STR4##
 3. The method of claim 2, wherein:(1) X represents N-tert-butyloxycarbonyl; (2) said esterifying step (A) comprises treating said compound 1' with pivaloyl chloride in dry pyridine; (3) said deprotecting step (S) comprises treating said compound 2' with p-toluenesulfonic acid in methanol; (4) said phosphorylating and hydrolyzing step (C) comprises treating said compound 3' with:(a) phosphorous oxychloride in the presence of triethylamine and CH₂ Cl₂, and then (b) 1N HCl in CHCl₃ ; and (5) said deprotecting step (D) comprises treating said compound 4' with:(a) tetrabutylammonium hydroxide in aqueous dioxane, AMBERLITE IR-120, water, and then (b) trifluoroacetic acid and CH₂ Cl₂.
 4. A method for chemically synthesizing N, N-dimethyl-sphingosine-1-phosphate comprising:(A) esterifying the C-3 hydroxyl group of compound 1' to give compound 2' ##STR5## wherein X represents a moiety that protects the amino group formed in step (B), (B) selectively deprotecting the C-1 hydroxyl group of said compound 2' to give compound 3' ##STR6## (C) eliminating the protecting moiety X from said compound 3' and then subjecting the product to reductive methylation to give compound 3^(a) ##STR7## (D) phosphylating the C-1 hydroxyl group of said compound 3^(a) followed by hydrolyzing to give compound 3^(b) ##STR8## (E) deprotecting the C-3 hydroxyl group to give compound 2, which is said N,N-dimethyl-sphingosine-1-phosphate ##STR9##
 5. The method of claim 4, wherein:(1) X represents N-tert-butyloxycarbonyl, (2) said esterifying step (A) comprises treating said compound 1' with pivaloyl chloride in dry pyridine; (3) said deprotecting step (B) comprises treating said compound 2' with p-toluenesulfonic acid in methanol; (4) said eliminating and reductive methylation step (C) comprises treating said compound 3' with:(a) trifluoroacetic acid in CH₂ Cl₂, and then (b) 37% CH₂ O and NaCNBH₃ in sodium acetate aqueous buffer; (5) said phosphylating and hydrolyzing step (D) comprises treating said compound 3^(a) with:(a) phosphorous oxychloride in the presence of triethanolamine and CH₂ Cl₂, and then (b) 1N HCl in CHCl₂ ; and (6) said deprotecting step (E) comprises treating said compound 3^(b) with tetrabutylammonium hydroxide in aqueous dioxane.
 6. A method for chemically synthesizing N,N,N-trimethyl-sphingosine-1-phosphate comprising:(A) esterifying the C-3 hydroxyl group of compound 1' to give compound 2' ##STR10## wherein X represents a moiety that protects the amino group formed in step (B), (B) selectively deprotecting the C-1 hydroxyl group of said compound 2' to give compound 3' ##STR11## (C) eliminating the protecting moiety X from said compound 3' and then subjecting the product to reductive methylation to give compound 3^(a) ##STR12## (D) permethylating said compound 3^(a) followed by treating with a basic anion exchange resin to give compound 3^(h) ##STR13## (E) phosphylating the primary hydroxyl group of said compound 3^(h) followed by hydrolyzing and then deprotecting the C-3 hydroxyl to give compound 3, which is said N,N,N-trimethyl-sphingosine-1-phosphate ##STR14##
 7. The method of claim 6, wherein:(1) X represents N-tert-butyloxycarbonyl, (2) said esterifying step (A) comprises treating said compound 1' with pivaloyl chloride in dry pyridine; (3) said deprotecting step (B) comprises treating said compound 2' with p-toluenesulfonic acid in methanol; (4) said eliminating and reductive methylation step (C) comprises treating said compound 3' with:(a) trifluoroacetic acid in CH₂ Cl₂, and then (b) 37% CH₂ O and NaCNBH₃ in sodium acetate aqueous buffer; (5) said permethylating step (D) comprises treating said compound 3^(a) with:(a) CH₃ I in NaHCO₃ and CHCl₃, and then (b) DOWEX 1×2 (Cl⁻), and (6) said phosphorylating, hydrolyzing, and deprotecting step (E) comprises treating said compound 3^(h) with:(a) POCl₃ in triethyl amine and CH₂ Cl₂, then (b) 1N HCl and CHCl₃, and then (c) tetrabutylammonium hydroxide in aqueous dioxane.
 8. A method for chemically synthesizing N-acyl and N-acetylsphingosine-1-phosphate comprising:(A) esterifying the C-3 hydroxyl group of compound 1' to give compound 2' ##STR15## wherein X represents a moiety that protects the amino group formed in step (B), (B) selectively deprotecting the C-1 hydroxyl group of said compound 2' to give compound 3' ##STR16## (C) eliminating the protecting moiety X, and then acylating or acetylating the unprotected amino group to give compound 3^(i) ##STR17## wherein Ac represents acyl or acetyl, (D) phosphylating the C-1 hydroxyl group of said compound 3^(i), followed by hydrolyzing to give compound 3^(j) ##STR18## (E) deprotecting the C-3 hydroxyl group to give compound 4, which is said N-acyl or N-acetylsphingosine-1-phosphate ##STR19##
 9. The method of claim 8, wherein:(1) X represents N-tert-butyloxycarbonyl; (2) said esterifying step (A) comprises treating said compound 1' with pivaloyl chloride in dry pyridene; (3) said deprotecting step (B) comprises treating said compound 2' with p-toluenesulfonic acid in methanol; (4) said eliminating and acylating or acetylating step (C) comprises treating said compound 3' with:(a) trifluoroacetic acid in CH₂ Cl₂, and then (b) CH₃ (CH₂)_(n) COCl, wherein n represents 0 to 22, in aqueous 55% K₂ CO₃ tetrahydrofuran; (5) said phosphylating and hydrolyzing step (D) comprises treating said compound 3^(i) with:(a) phosphorous oxychloride in the presence of triethylamine and CH₂ Cl₂, and then (b) 1N HCl in CHCl₃ ; and (6) said deprotecting step (E) comprises treating said compound 3^(j) with tetrabutylammonium hydroxide in dioxane. 